Regulation of hair cell and stomatal size by a hair-cell specific peroxidase in the grass Brachypodium distachyon

The leaf epidermis is the outermost cell layer forming the interface between plants and the atmosphere that must both provide a robust barrier against (a)biotic stressors and facilitate carbon dioxide uptake and leaf transpiration 1. To achieve these opposing requirements, the plant epidermis developed a wide range of specialized cell types such as stomata and hair cells. While factors forming these individual cell types are known 2–5, it is poorly understood how their number and size is coordinated. Here, we identified a role for BdPRX76/BdPOX, a class III peroxidase, in regulating hair cell and stomatal size in the model grass Brachypodium distachyon. In bdpox mutants prickle hair cells were smaller and stomata were longer. Because stomatal density remained unchanged, the negative correlation between stomatal size and density was disrupted in bdpox and resulted in higher stomatal conductance and lower intrinsic water-use efficiency. BdPOX was exclusively expressed in hair cells suggesting that BdPOX cell-autonomously promotes hair cell size and indirectly restricts stomatal length. Cell wall autofluorescence and lignin stainings indicated a role for BdPOX in lignification or crosslinking of related phenolic compounds at the hair cell base. Ectopic expression of BdPOX in the stomatal lineage increased phenolic autofluorescence in guard cell walls and restricted stomatal elongation in bdpox. Together, we highlight a developmental interplay between hair cells and stomata that optimizes epidermal functionality. We propose that cell-type-specific changes disrupt this interplay and lead to compensatory developmental defects in other epidermal cell types.

In the model grass B. distachyon, the leaf blade epidermis is dominated by rectangular pavement cells, stomatal complexes, trichomes consisting of prickle hair cells (PHCs) and few interspersed macrohairs, and occasional silica cells (Fig. 1A). Most specialized amongst epidermal cells are stomatal complexes, which are cellular pores on the epidermis that drive plant gas exchange. Fast stomatal movements and adjustments in stomatal anatomy (stomatal density and size) are crucial for water-use efficient gas exchange thereby contributing to abiotic stress resilience 6,7 . Yet, since stomatal density and size are negatively correlated, it remains elusive how modifying just a single of these anatomical traits affects stomatal function.
To identify novel factors associated with graminoid stomatal morphology and functionality, we performed RNA-sequencing of mature zones of 7 day old leaves in B. distachyon Bd21-3 (WT) and bdmute plants (Fig. S1A). The bdmute leaf epidermis features abnormal stomata that lack SCs, which strongly affects stomatal responsiveness and gas exchange 3 . 179 genes were downregulated in bdmute (Supplementary Dataset) and we selected ~50 candidate genes for a reverse genetic screening. Candidates were chosen according to their annotated gene function, their expression being lower in the developmental zone 8 and the availability of mutants from a collection of sodium azide (NaN 3 ) mutagenized and fully resequenced lines 9 .
In the initial screen, we found that two mutants of the class III peroxidase BdPRX76/BdPOX (BdiBd21-3.2G0467800;

Regulation of hair cell and stomatal size by a hair-cell specific peroxidase in the grass Brachypodium distachyon
The leaf epidermis is the outermost cell layer forming the interface between plants and the atmosphere that must both provide a robust barrier against (a)biotic stressors and facilitate carbon dioxide uptake and leaf transpiration 1 . To achieve these opposing requirements, the plant epidermis developed a wide range of specialized cell types such as stomata and hair cells. While factors forming these individual cell types are known 2-5 , it is poorly understood how their number and size is coordinated. Here, we identified a role for BdPRX76/BdPOX, a class III peroxidase, in regulating hair cell and stomatal size in the model grass Brachypodium distachyon. In bdpox mutants prickle hair cells were smaller and stomata were longer. Because stomatal density remained unchanged, the negative correlation between stomatal size and density was disrupted in bdpox and resulted in higher stomatal conductance and lower intrinsic water-use efficiency. BdPOX was exclusively expressed in hair cells suggesting that BdPOX cell-autonomously promotes hair cell size and indirectly restricts stomatal length. Cell wall autofluorescence and lignin stainings indicated a role for BdPOX in lignification or crosslinking of related phenolic compounds at the hair cell base. Ectopic expression of BdPOX in the stomatal lineage increased phenolic autofluorescence in guard cell walls and restricted stomatal elongation in bdpox. Together, we highlight a developmental interplay between hair cells and stomata that optimizes epidermal functionality. We propose that cell-type-specific changes disrupt this interplay and lead to compensatory developmental defects in other epidermal cell types. .2G0467800) gene model indicating the location and nature of the mutations in bdpox-1 (NaN1508) and bdpox-2 (NaN1528). (C) Stomatal conductance (g sw ) in response to changing light (1000-100-1000-0 PAR) in WT, NaN1508 BdPOX , bdpox-1 and bdpox-2 (n=6 individuals per genotype). Dots represent the mean and error bars represent SEM. p value obtained from one-way ANOVA comparing differences on mean g sw among different groups indicated in the graph. Full statistical analysis demonstrating significant differences between wild-type and bdpox mutants in Supplementary dataset. (D) Intrinsic water-use efficiency (iWUE) in response to changing light (1000-100-1000 PAR) in WT, NaN1508 BdPOX , bdpox-1 and bdpox-2 (n=6 individuals per genotype). Dots represent the mean and error bars represent SEM; p-value obtained from one-way ANOVA comparing differences on mean g sw among different groups is indicated in the graph. Full statistical analysis demonstrating significant differences between wild-type and bdpox mutants can be found in Fig. 1B; S1A, B) showed lower intrinsic water-use efficiency (iWUE; Fig. S1C) and higher ambient-adapted stomatal conductance (g sw ; Fig. S1D). The two NaN mutants disrupting BdPRX76/BdPOX were NaN1508, which contained a heterozygous, early STOP codon (E44*; bdpox-1) and NaN1528, which contained a homozygous missense mutation in the BdPOX active/heme-binding site (A197S; bdpox-2; Fig. 1B, S1B). From the segregating NaN1508 population, homozygous mutant individuals (bdpox-1, NaN1508 bdpox-1 ) and wildtype segregants (NaN1508 BdPOX ) were selected by genotyping. Because NaN1508 BdPOX contained the same background mutations as bdpox-1, we included it as an additional wild-type control line.
To confirm the gas exchange defects in bdpox mutants, we measured stomatal conductance (g sw ) in response to changing light intensity (1000-100-1000-0 PAR) 7 . In bdpox plants, we observed higher g sw in all light steps (Fig. 1C), but no significant impact on stomatal opening and closure speed ( Fig.  S2A-C). Since no significant variation in carbon assimilation (A) was observed (Fig. S2D), bdpox mutants suffered a decrease in intrinsic water-use efficiency (iWUE, A/g sw ), particularly in the light-limited step (100 PAR; Fig. 1D).
To test if the increased g sw was caused by changes in stomatal density we performed microscopic analysis of the leaf epidermis from the leaves assessed for gas exchange. No differences were found regarding stomatal density (SD; Fig. 1E), yet we observed significantly longer stomata in bdpox mutants (Fig. 1F). This suggested that the well-established negative correlation between stomatal size and density, observed both interspecifically 10-15 and intraspecifically 7,16,17 , was disrupted in bdpox (Fig. 1G). Detailed morphometric confocal analysis of fusicoccin-treated leaves to induce full stomatal opening revealed that stomatal pores are indeed longer and larger in bdpox ( Fig. S2F-I).
To test whether the higher g sw levels could be explained by the disrupted stomatal anatomy in bdpox, we compared physiological g s max measurements with anatomical g s max calculations (theoretical g s max based on gas diffusion physical constants and stomatal anatomical traits) using an established equation recently optimized for grass stomata 7 . Physiological g s max measurements confirmed the increased g sw capacity in bdpox mutants (Fig. 1H), and anatomical g s max calculations revealed the same relative variation between bdpox mutants and WT (Fig. 1I). Together, this strongly suggested a causal relationship between longer stomata and higher gas exchange in bdpox.
To verify if the cell size defect was specific to stomata, we also measured the length of pavement cells (PCs) and of the base of prickle hair cells (PHCs). While no differences were found in PC size among genotypes (Fig. S2J), we observed an unexpected decrease in the base length of PHCs in bdpox mutants (Fig. 1J).
In summary, BdPOX seemed to negatively affect stomatal size but positively regulate PHC size.

BdPOX is expressed in hair cells and mutant complementation rescued stomatal and prickle hair cell phenotypes
To determine where BdPOX was expressed, we generated transcriptional (BdPOXp:3xNLS-eGFP) and translational (BdPOXp:BdPOX-mCitrine) reporter lines. To our surprise, both BdPOX reporter genes were exclusively expressed in PHCs ( Fig. 2A, B; S3B, Movie S1) and macrohairs (Fig. S3A). Because grass leaf development and, consequently, epidermal development follows a strict base-to-tip developmental gradient with well-established stomatal stages, we used the stomatal stages as landmarks to track PHC development 18 . We observed that both transcriptional (BdPOXp:3xNLS-eGFP) and translational (BdPOXp:BdPOX-mCitrine) reporter expression in PHCs started during stages 5-6i of stomatal development and, therefore, before significant stomatal elongation ( Fig. 2A, B).
(g sw ) was restored to wild-type levels in the complementation lines (Fig. 2F).
Ubiquitous expression of BdPOX in bdpox1, however, had no effect on cell size nor on gas exchange suggesting that a cell type-specific expression is necessary for its complementation ( Fig. S3F-I). ZmUbip-driven BdPOX was also expressed at significantly lower levels in PHCs (0.5 fold decrease) compared to BdPOX driven by its endogenous promoter in the BdPOXp:BdPOX-mCitrine line (Fig. S3F, J, K). Therefore, not only correct spatiotemporal expression but also correct dosage might be required to complement the mutant phenotype.
Together, our results suggested that BdPOX played a cell-autonomous role in promoting PHC size and, as a consequence, indirectly restricted stomatal elongation.

BdPOX might be involved in lignification/hydroxycinnamates cross-linking at the base of prickle hair cells to positively regulate cell outgrowth
To mechanistically link how a hair-cell localized class III peroxidase could affect stomatal anatomy, we first investigated the cell-autonomous function of BdPOX in PHCs. BdPRX76/BdPOX seems to be in a monocot-specific clade 19 , yet its closest Arabidopsis homolog, AtPRX66, is associated with phenolic modifications in the cell wall, namely lignification of tracheary elements 20 . Class III peroxidases can function through a hydroxylic cycle consuming H 2 O 2 or through a peroxidative cycle, producing H 2 O 2 and participating in the polymerization and crosslinking of phenolic compounds (including monolignols into lignins) [21][22][23][24] . A unique feature of grass cell walls is the significant yet cell-type-specific amount of hydroxycinnamates (ferulic acid and ρ-coumaric acids) that are bound to arabinoxylans and/or to lignins [25][26][27][28] . Thus, we hypothesized that BdPOX modulates PHC size by altering phenolic compounds in the cell walls.
To test this, we assessed UV-induced autofluorescence of phenolic compounds in the cell wall of PHCs [29][30][31] . PHC autofluorescence plot profiles and corrected total cell fluorescence revealed lower autofluorescence in bdpox-1 compared to WT and complemented bdpox-1, specifically at the base of the PHCs (approximately the initial 12 µm; Fig. 3A To test if the lower cell wall autofluorescence originated from decreased lignin content, we used different histochemical stainings. Basic fuchsin is a standard lignin stain 32-34 that also has a high affinity for hydroxycinnamates in the B. distachyon cell wall 35 . Simultaneous imaging of cell wall autofluorescence and fuchsin-stained lignin showed that fuchsin preferentially stained the lower section of PHCs while total phenolics autofluorescence was observed until the tip (Fig. 3D-F; Movie S2, Movie S3). Indeed, bdpox-1 mutants showed lower fuchsin fluorescence intensity at the basal section of PHCs (first 12 µm from the basal outline) compared to WT and complemented bdpox-1 lines (Fig. 3E, F) suggesting re-duced lignin/hydroxycinnamates content in the mutant. Very similar results were observed using safranin-O lignin staining (Fig. S4B, C), in which increased red fluorescence is observed in lignified cells, whereas non-lignified cell walls preferentially fluoresce in green 36 . Therefore, a red/green ratiometric analysis allows for a semi-quantitative evaluation of cell wall lignification 37 . bdpox-1 mutants displayed a lower safranin-O ratio at the base section of PHCs (first 12 µm) compared to WT and complemented bdpox-1, again indicating a decrease in lignin content in the mutant (Fig. S4B, C).
Regarding stomata, no significant differences were observed in autofluorescence nor in fuchsin-stained lignin in mature GCs between WT and bdpox-1 (Fig. S4D, E). When looking at the developing stomata during stomatal elongation/maturation, we observed that cell wall autofluorescence increased in wild-type GCs (Fig. S4F). This increase appeared to start earlier in bdpox-1 but stalled sooner, too, which may be linked to the stomatal elongation defects in the mutants (Fig. S4F).
Overall, our data suggests that BdPOX participates in lignification of the basal cell wall of PHCs, which seems to be required for proper PHC growth, and indirectly impacts stomatal elongation.

Specific expression of BdPOX in the stomatal lineage arrests stomatal elongation
BdPOX appeared to be involved in cell wall phenolic modifications (lignification/crosslinking) at the base of PHCs to promote cell outgrowth. How this PHC-specific process, however, affected stomatal elongation remained elusive. When following the basipetal developmental gradient of the B. distachyon epidermis, we found that PHCs grow and mature significantly before the stomatal complexes start to elongate (Fig. 4A). PHC outgrowth started when stomata were in early stages of development (i.e. stage 3-4 during SC recruitment) and was completed before GCs elongated and acquired the mature dumbbell morphology (Fig. 4A). Therefore, the growth restriction of PHCs in bdpox could secondarily influence stomatal anatomy, but not vice versa. Accordingly, the ectopic expression of BdPOX in the developing GC lineage using a stomatal-lineage specific promoter (BdMUTEp:Bd-POX(CDS)-mCitrine; Fig. 4B) significantly restricted stomatal elongation in bdpox-1, which correlated with increased phenolics autofluorescence in GCs (Fig. 4C, D) but not in fuchsin staining as observed in PHCs (Fig. S4G). Therefore, the ectopic expression of BdPOX in GCs seems to affect different polyphenolic compounds than lignin/hydroxycinnamates, which restricts excessive GC elongation in bdpox mutants (Fig.  4D). PHC size, however, remained unaffected (Fig. 4E) as PHCs mature before stomata elongate.
Intriguingly, we observed aberrant cell divisions in the pavement cells surrounding stomata when expressing Bd-MUTEp:BdPOX(CDS)-mCitrine in bdpox-1 (Fig. 4F, G). This suggested that elongating stomata might compensate for tissue-wide mechanical imbalances caused by too small PHCs in the bdpox-1 epidermis. Thus, when ectopic, stomatal lineage-specific expression of BdPOX inhibited the compensatory stomatal elongation in bdpox-1, additional divisions in pavement cells around stomata might be triggered to compensate for these tissue-wide mechanical tensions instead.
In conclusion, ectopically expressed BdPOX in GCs arrested the excessive stomatal elongation in bdpox-1, potentially by modifying phenolic cell wall components distinct from lignin/hydroxycinnamates. Furthermore, the observation of ectopic pavement cell divisions around length-restricted stomata in bdpox suggests that a growth disruption in one epidermal cell-type leads to compensatory developmental defects in other epidermal cells.

Conclusions
The leaf epidermis is the barrier between the inner photosynthetically active tissues and the environment. Highly specialized epidermal cell types like stomata and hair cells facilitate the contrasting functional requirements of this outermost barrier. Several studies suggest that hair cell patterning intersects with the core stomatal developmental programs in Arabidopsis [38][39][40][41] . Also in grasses, failing to specify stomatal identity results in hair cells being formed in their place in B. distachyon 2 and failing to specify hair cell identity leads to ectopic stomata in maize 5 . This suggests that these cell types are ontogenetically closely related in grasses and that their development is thus likely coordinated. Furthermore, stomatal and trichome densities were shown to be negatively correlated in Solanum lycopersicum. The stomata to trichome ratio determined water-use efficiency 42 suggesting a physiological relevance for the coordination between the two cell types. Yet, the mechanisms that coordinate the formation and growth of stomata and trichomes remain highly unexplored. Many core players guiding grass stomatal development were characterized 2,3,18,43-50 , whereas grass hair cell formation remains poorly explored 41 . Few grass trichome initiation factors such as the transcription factors HAIRY LEAF 6 (HL6) [51][52][53] and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 10/14/26 (SPL10/14/26) 5,54,55 were identified in rice and maize, but factors that affect morphogenesis and size remain mostly unknown. Here, we identified BdPOX and revealed its role in regulating PHC size, which indirectly affects stomatal size and optimal water-use efficiency in the model grass B. distachyon. BdPOX was exclusively expressed in the hair cells and seemed to participate in the lignification or/ and cross-linking of cell wall phenolics (such as hydroxycinnamates) specifically at the base of PHCs. Since lignin is a cell wall polymer that provides mechanical support 56-58 we speculate that such cell wall modifications at the base of PHCs are required to increase tensile strength and provide physical support for cell outgrowth. Similar processes are required in tip-growing root hairs and pollen tubes. In both cases, mechanical anisotropy along the main growth axis, which is mediated by modifying cell wall properties, seems to support cell outgrowth [59][60][61] . Sustained perpendicular PHC outgrow might then feedback on PHC base expansion to maintain optimal geometrical proportions of these cells. When BdPOX was misexpressed in bdpox GCs, cellular growth was restricted rather than promoted. This differential effect on cell elongation likely has several reasons; first, different polyphenolic compounds were affected in the GC context compared to the PHC context. This might have distinct effects on cell wall properties and cellular mechanics. Second and unlike in PHCs, ectopic BdPOX expression in the GC context occurs much before cell elongation commences potentially leading to premature cell wall stiffening and, thus, growth restriction. Third, PHCs grow perpendicular rather than parallel to the principal direction of leaf growth like GCs. Consequently, (localized) modification of cell wall properties might affect growth differently in tip-growing versus non-tip-growing cells.
The reduction in PHC size in bdpox indirectly altered stomatal size, but did not translate to changes in stomatal density. The resulting disruption of the negative correlation between stomatal size and density likely has two reasons; first, changes to stomatal size occur much later in development than the determination of stomatal density. Thus, changes in stomatal density can affect stomatal size a posteriori, where an increase in stomatal numbers can induce a downstream effect on the cell-wall machinery controlling stomatal elongation. This process is very unlikely to happen in the other direction particularly in grasses, where early stages are not only temporally but also spatially separated from late stages. Second, bdpox primarily impacts the PHC lineage and only affects stomatal development as a secondary consequence. Without a disruption of the stomatal genetic toolbox itself a compensatory mechanism might not be induced in a timely manner.
However, the exact mechanism of how restricting PHC growth induces stomatal elongation remains vague. We speculate that decreased PHC size may lead to changes in mechanical and/or geometrical constraints in the epidermal tissue, which would allow for increased stomatal elongation as a compensatory mechanism to reconstitute the tensile balance in the epidermis. The increase in stomatal length observed in bdpox mutants (~9 %) was quantitatively equivalent to the decrease in PHC base length (~10 %). In addition, expressing BdPOX in the GC lineage of bdpox-1 resulted in an epidermis containing both shorter stomata and shorter PHCs and induced aberrant cell divisions surrounding the stomatal complexes. We speculate that the combination of restricted PHC growth due to bdpox-1 and a prevention of stomatal elongation due to GC-expressed BdPOX may have caused a mechanical imbalance in the elongating epidermis resulting in cell divisions to compensate cellular tensions particularly around stomatal complexes. Alternatively, changes in hydrogen peroxide levels in the PHC apoplast due to loss of BdPOX might affect the reactive oxygen species signaling landscape, which could influence stomatal length non-cell-autonomously.
Regardless, the unique disruption in the negative correlation between stomatal size and density allowed us to in-vestigate how modifying a single stomatal anatomical trait (i.e. stomatal size) would affect gas exchange. While an increase in stomatal size enhanced stomatal conductance, it did not significantly affect stomatal opening and closing speed, corroborating our previous observation that stomatal speed was correlated with stomatal density but not with stomatal size in B. distachyon 7 .
Overall, we identified a hair cell-specific factor and demonstrated how a cell-type-specific disruption of PHC growth indirectly affected stomatal development. Strikingly, this indirect effect allowed for the specific manipulation of stomatal size without affecting stomatal density. This enabled us to test how the manipulation of a single stomatal anatomy trait (i.e. stomatal size) affected stomatal gas exchange physiology and water-use efficiency in a grass model for the first time. Consequently, manipulating PHC size might present an indirect route to potentially alter stomatal size in grasses without affecting stomatal density.

DECLARATION OF INTERESTS
The authors declare no competing interests.

DATA AND CODE AVAILABILITY
RNA-seq data have been deposited at GEO and are publicly available (GEO accession number GSE206682). All quantitative data generated and analyzed in this study can be found in the Supplementary Dataset. Microscopy images reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

SUPPLEMENTARY INFORMATION
Supplemental Information includes four figures, three movies, and one supplemental dataset file and can be found online at https://figshare.com/projects/Supplementary_In-formation_for_Nunes_et_al_2023_-_BdPOX/163402

RNA-sequencing
25 mature leaf zones (25-30 mm from the base of 2 nd leaves) per replicate were collected from three wild-type (Bd21-3) replicates and from three bdmute replicates (7 days after germination seedlings grown on ½ MS plates at 20 o C with ~100 µmol photons m -2 s -1 light) were carefully collected, snap-frozen in liquid nitrogen and grounded using mortar and pistil. RNA extraction, library preparation, RNA-sequencing and data analysis was essentially performed as described in Zhang et al. (2022) 8 . To be specific, total RNA was isolated using Qiagen's RNeasy Plant Mini kit with on-column DNAse digestion according to the manufacturer's instructions. The Kapa mRNA HyperPrep (Roche) was used to generate an mRNA enriched sequencing library with an input of 1µg of total RNA. The libraries were sequenced using the Illumina NextSeq500 platform. Read quality was assessed with FastQC and mapped against the Bd21-3v1.0 genome using bowtie2. Mapped reads were counted using summarized overlap and differentially expressed genes were analyzed using DeSeq2. Finally, gene expression was normalized by transcripts per kilobase million (TPM). Raw and processed data are available at Gene Expression Omnibus (GEO) with the accession number GSE206682.

Reporter Constructs
Reporter and overexpression constructs were generated using the Greengate cloning system 63 . BdPOX promoter and coding sequences were amplified from wild-type Brachypodium distachyon (Bd21-3) genomic DNA extracted using a standard CTAB DNA extraction protocol 68  To clone the BdPOX genomic sequence, a point mutation was induced in the genomic BdPOX sequence to eliminate a BsaI/Eco31I site (GGTCTC) in the second intron. Genomic BdPOX was amplified in two separate fragments using priTN99/priTN102 and priTN100/priTN101 (containing the bp substitution AGTCTC). The two resulting PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Ref. REF 740609.50; Macherey-Nagel, Düren, Germany), digested at 37 o C overnight using FastDigest Eco31I (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and ligated overnight at 16 o C using T4 ligase (NEB, Ipswich, Massachusetts, USA). The fully reassembled BdPOX gene was ligated overnight at 16 o C with the previously digested (FastDigest Eco31I) and dephosphorylated pGGC000 entry vector (with Antarctic Phosphatase, NEB) to generate pGGC_BdPOX. BdPOX (CDS) sequence was amplified using priTN99/priTN100 from cDNA. The resulting PCR product was purified and digested using FastDigest Eco31I (Thermo Fisher Scientific), and ligated using T4 ligase (NEB) with previously digested (FastDigest Eco31I) and dephosphorylated (Antarctic Phosphatase) pGGC000 entry vector to generate pGGC_BdPOX(CDS).

BdPOXp:BdPOX-mCitrine, ZmUbip:BdPOX:mVenus, ZmUbip:BdPOX(CDS)-mCitrine, BdMUTEp:BdPOX(CDS)-mCitrine and
BdPOXp:3xNLS-eGFP were generated using the Green Gate assembly 63 . In short, the 6 different entry modules (pGGA_ specific promoter; pGGB_dummy or N-tag; pGGC_gene sequence or tag; pGGD_dummy or C-tag; pGGE_terminator and pGGF_resistance) were repeatedly digested and ligated with the destination vector pGGZ004 during 50 cycles (5 min 37 o C followed by 5 min 16 o C) followed by 5 min at 50 o C and 5 min at 80 o C for heat inactivation of the enzymes. All final constructs were test digested and the generated GreenGate overhangs were Sanger sequenced.

Generation and Analysis of Transgenic Lines
Embryonic calli derived from Bd21-3 and bdpox-1 parental plants were transformed with AGL1 Agrobacterium tumefaciens containing the binary expression vectors, selected based on hygromycin resistance, and regenerated as described in Zhang et al. (2022) 8 . In short, young, transparent embryos were isolated and grown for three weeks on callus induction media (CIM; per L: 4.43g Linsmaier & Skoog basal media (LS; Duchefa #L0230), 30g sucrose, 600µl CuSO4 (1mg/ ml, Sigma/Merck #C3036), 500µl 2,4-D (5mg/ml in 1M KOH, Sigma/Merck #D7299), pH 5.8, plus 2.1g of Phytagel (Sigma/Merck #P8169)). After three weeks of incubation at 28 o C in the dark, crisp, yellow callus pieces were subcultured to fresh CIM plates and incubated for two more weeks at 28 o C in the dark. After two weeks, calli were broken down to 2-5mm small pieces and subcultured for one more week at 28 o C in the dark. For transformation, AGL1 Agrobacteria with the desired construct were dissolved in liquid CIM media (same media as above without the phytagel) with freshly added 2,4-D (2.5µg/ml final conc.), Acetosyringone (200µM final conc., Sigma/Merck #D134406), and Synperonic PE/ F68 (0.1% final conc., Sigma/Merck #81112). The OD600 of the Agrobacteria solution was adjusted to 0.6. Around 100 calli were incubated for at least 10 min in the Agrobacteria solution, dried off on sterile filter paper and incubated for three days at room temperature in the dark. After three days, transformed calli were moved to selection media (CIM + Hygromycin (40µg/ ml final conc., Roche #10843555001) + Timentin (200µg/ ml final conc., Ticarcillin 2NA & Clavulanate Potassium from Duchefa #T0190)) and incubated for one week at 28ºC in the dark. After one week, calli were moved to fresh selection plates and incubated for two more weeks at 28ºC in the dark. Next, calli were moved to callus regeneration media (CRM; per L: 4.43g of LS, 30g maltose (Sigma/Merck #M5885), 600µl CuSO4 (1mg/ml), pH 5.8, plus 2.1g of Phytagel). After autoclaving, cool down and add Timentin (200µg/ml final conc.), Hygromycin (40µg/ml final conc.), and sterile Kinetin solution (0.2µg/ml final conc., Sig-ma/Merck #K3253). Calli were incubated at 28 o C and a 16h light:8h dark cycle (70-80 μmol PAR m −2 s -1 ). After 1-6 weeks in the light, shoots will form. Move shoots that are longer than 1cm and ideally have two or more leaves, to rooting cups (Duchefa #S1686) containing rooting media (per L: 4.3g Murashige & Skoog including vitamins (Duchefa #M0222), 30g sucrose, adjust pH to 5.8, add 2.1g Phytagel). After autoclaving, cool down and add Timentin (200µg/ml final concentration). Once roots have formed, plantlets can be moved to soil (consisting of 4 parts ED CL73 (Einheitserde) and 1 part Vermiculite) and grown in a greenhouse with 18h light:6h dark cycle (250-350 μmol PAR m −2 s -1 ). Ideally, the transgenic plantlets moved to soil are initially kept at lower temperatures (day temperature = 22 o C, night temperature = 18-20 o C) for 2-4 weeks until they have rooted sufficiently before being moved to the warmer greenhouse (day temperature = 28 o C, night temperature = 22 o C).
We refer to Brachypodium regenerants as T0 and to the first segregating population as T1. We analyzed 5-10 independent lines in the T0 generation (depending on how many independent lines were recovered upon regeneration). We confirmed the observed expression pattern and performed the phenotyping studies using one to three independent and fertile T0 transgenics that produced seeds (3-4 T1 individuals per line).

Gas Exchange Phenotyping
Infra-red gas analyser-based leaf-level gas exchange measurements were performed as described in Nunes et al. (2022) 7 . All measurements were performed on the youngest fully expanded mature leaf of B. distachyon plants 3 weeks after sowing (17-24 days after sowing) using a LI-6800 (LI-COR Biosciences Inc, Lincoln, NE, USA). Ambient light intensity was monitored during the measurements using an external LI-190R PAR Sensor attached to LI-6800. Greenhouse temperature and relative humidity were monitored during the experiments using an Onset HOBO U12-O12 4-channel data logger that was placed next to the plants used for analysis.
Light response kinetics: LI-6800 chamber conditions were as follows: flow rate, 500 µmol s -1 ; fan speed, 10000 rpm; leaf temperature, 28 o C; relative humidity (RH), 40 %; [CO 2 ], 400 µmol mol -1 ; photosynthetic active radiation (PAR), 1000 -100 -1000 -0 µmol PAR m -2 s -1 (20 min per light step). Gas exchange measurements were automatically logged every minute. The leaf section measured inside the LI-6800 chamber was collected, fixed and cleared to measure leaf area and accurately determine A and g sw and stomatal anatomical parameters like stomatal length and density. iWUE was calculated as the ratio of A to g sw . Stomatal opening and closure speed were evaluated by rate constants (k, min -1 ) determined from exponential equations fitted for each of the three light transitions (1000-100, 100-1000 and 1000-0 PAR), as described in Nunes et al. (2022) 7 .
Anatomical g s max calculations: Anatomical g s max calculations were performed on the 6 individuals for which gas exchange and stomatal anatomy was assessed (Fig. 1C-H). Based on the formula optimized for B. distachyon in   7 this requires the measurement of three anatomical parameters: stomatal length, stomatal density and GC width at the apex (average of 30 stomata per individual) from images obtained using a Leica DM5000B microscope.
Steady-state stomatal conductance: LI-6800 chamber conditions were as follows: flow rate, 500 µmol s -1 ; fan speed, 10000 rpm; leaf temperature, 28 o C; relative humidity (RH), 40 %; [CO 2 ], 400 µmol mol -1 ; photosynthetic active radiation (PAR), 1000 PAR m -2 s -1 (20 min). Gas exchange measurements were automatically logged every minute. The leaf section measured inside the Li-6800 chamber was collected to measure leaf area to accurately determine A and g sw and, then fixed and cleared to determine stomatal anatomical parameters like stomatal length and density. All measurements were performed in a semi-randomized manner between 11:30 and 17:30 h to assure measurements for each genotype covered identical periods of time of the day and to avoid the influence of the diurnal variation of g sw observed in Nunes et al. (2022) 7 .
Ambient-adapted stomatal conductance: Steady-state ambient-adapted stomatal conductance was assessed using a SC-1 porometer (Meter, Pullman, Washington, USA). SC-1 was calibrated using the calibration plate and the moist circular filter paper provided with the SC-1. Each measurement was performed in automode (30 s). The relative humidity of the SC-1 porometer was returned to < 10 % after each measurement by shaking the sensor head for 30-90 s. Three to four fully expanded leaves per individual were measured twice. Three B. distachyon (Bd21-3) and three bdpox-1 individuals were assessed three weeks after sowing. All measurements were performed in a randomized manner between 8:00 and 9:30 h, to avoid the influence of the diurnal variation of g sw observed in Nunes et al. (2022) 7 .
Important note: Some of the wild-type gas exchange measurements were previously published in Nunes et al. (2022) 7 , where 120 wild-type measurements over the course of 2 years were correlated to variable growth conditions. The WT gas exchange data that was published in Nunes et al. (2022) 7 is indicated accordingly in the Supplementary Dataset.

Microscopy and Phenotypic Analysis
Most of the morphometric and the cell wall measurements were performed on the actual leaf segments used for gas exchange measurement to thoroughly link cellular form and composition to stomatal gas exchange.
Leaf epidermis morphometry: For DIC imaging, the youngest fully expanded mature leaves (3 weeks after sowing) were collected after LI-6800 measurements and placed into 7:1 ethanol:acetic acid and incubated overnight to fix the leaf tissue and remove chlorophyll. To prepare samples for imaging, the tissue was rinsed twice in water, mounted on slides in Hoyer's solution 69 and the abaxial side was examined using a Leica DM5000B microscope (Leica Microsystems, Wetzlar, Germany). Typically, 4-6 (40x objective) and 3-5 (20x objective) abaxial fields of view per leaf of each individual plant were imaged to determine stomatal length, stomatal density, stomatal width at the apices, pavement cell length, prickle hair cell (PHC) base length, base area and/or PHC outgrowth using the straight line tool xyz and/or the polygon selection tool in Fiji 66 . In the case of complementation experiments represented in Figure 2, the slides were prepared and randomized by an independent researcher before measurements on Fiji, to avoid potential biased phenotyping. The confocal morphometrical analysis of stomata in Bd21-3 and bdpox-1 mutants, were performed as described in Nunes et al. (2022) 7 . Leaves were incubated overnight in buffer solution (50mM KCl, 10mM MES-KOH) with 4 mM Fusicoccin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; Cat. no. 20108-  in the light to force stomatal opening. Leaves were stained in propidium iodide (Sigma-Aldrich, St. Louis, Missouri, USA, Cat. no. P3566; PI, 1:100 of a 1 mg/ml stock) for 5 min and Z-stacks were taken using confocal microscopy. Image analysis was done using Fiji to measure stomatal pore length and stomatal pore area (hand-traced).
Stomatal associated-epidermal defects: Stomata and stomata surrounded by defective cell divisions were counted in 5 abaxial fields of view (40 x objective) per leaf of each individual plant. Finally the percentage of stomatal-associated defects was calculated as the total number of stomata surrounded by defective cell divisions using the following formula: sum of 5-7 fields of view)/total number of stomata (sum of 5-7 fields of view) x 100.
Reporter lines: For confocal imaging, emerging 2 nd (6-7 days post germination (dpg)) or 3 rd (11-12 dpg) leaves from plants grown on plates were carefully pulled from the sheath of the older leaf to isolate and reveal the developmental leaf zone. Samples were stained in propidium iodide (PI, 1:100 of a 1 mg/ml stock) for 5 min to stain cell walls and/or mounted directly in water for imaging on a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany). Image analysis was done using Fiji.
Total phenolics autofluorescence: Small leaf fragments previously fixed and cleared in 7:1 ethanol:acetic acid were washed in 70 % ethanol and then transferred to distilled water with 0.02 % (v/v) Tween for rehydration for 3 hours and mounted in distilled water for imaging. Samples were imaged on a Leica SP8 confocal microscope. Excitation and detection settings were as follows: Ex. 405 nm and Em. 490-550 nm. Laser power set to 10 %. For the analysis of PHCs, stacks of 0.33 μm steps were obtained and plot profile analysis was performed on sum slices Z-projections in Fiji. For the analysis of PHCs plot profiles, a straight line was drawn from the base of the PHCs to the tip and plot profiles of gray values were obtained. For the analysis of PHC average autofluorescence, PHCs were handtraced on the sum slices Z-projections and CTCF (corrected total cell fluorescence) calculated as Integrated Density -(Area of selected cell x Mean fluorescence of background readings). Mean fluorescence of 9 background readings per image were obtained to calculate CTCF (using the traced area at regions with only background signal) and to correct plot profiles. For the analysis of mature stomata autofluorescence, GCs were handtraced and autofluorescence was calculated as CTCF (corrected total cell fluorescence) = Integrated Density -(Area of selected cell x Mean fluorescence of background readings) from single images. Mean fluorescence of 3-4 background readings per image were obtained to calculate CTCF (using the traced area at regions with only background signal).
For the analysis of phenolic compounds during stomatal elongation (step 6i-6iii), the emerging 2 nd (6-7 dpg) or 3 rd (11-12 dpg) leaves were carefully pulled from the sheath of the older leaf to isolate and reveal the developmental leaf zone were mounted in water for imaging on a Leica SP8 confocal microscope. Autofluorescence intensity was measured on handtraced GCs from sum slices Z-projections (75 stacks) using Fiji and calculated as CTCF (corrected total cell fluorescence) = Integrated Density -(Area of selected cell x Mean fluorescence of background readings). Mean fluorescence of 3-4 background readings surrounding each stoma were obtained to calculate CTCF for each GC pair.
Basic fuchsin staining: Small leaf fragments previously fixed and cleared in 7:1 ethanol:acetic acid were transferred to distilled water with 0.02 % (v/v) Tween for rehydration for 3 hours. Samples were incubated in 30 µl of 0.01 % Basic Fuchsin (Sigma-Aldrich, St. Louis, Missouri, USA, Cat. no. 857343) for 5 min and washed twice with 30 µl of 50 % glycerol (v/v) for 5 min (2.5 min per wash step), and mounted in 50 % glycerol. Samples were imaged under Ex. 561 nm and Em. 573-603 nm. Stacks of 0.33 μm steps were obtained. For the analysis of PHCs, sum slices Z-projections were performed. A straight line was drawn from the base of the PHCs until the tip and plot profiles of gray values were obtained. Mean gray value of 9 background readings was obtained to correct each measurement. For the analysis of stomata, GCs were handtraced and fluorescence was calculated as CTCF (corrected total cell fluorescence) = Integrated Density -(Area of selected cell x Mean fluorescence of background readings) from single images. Mean fluorescence of 3-6 background readings was obtained for each image to calculate CTCF.
Safranin-O staining: Small leaf fragments previously fixed and cleared in 7:1 ethanol acetic acid were transferred to distilled water for 2 hours. Samples were incubated in 50 µl of 0.2 % Safranin-O (Sigma-Aldrich, St. Louis, Missouri, USA, Cat. no. 84120; v/v in 50 % EtOH) for 10 min, washed with 50 % EtOH for 10 min and hydrated in distilled water for 15 min. Samples were mounted in distilled water and imaged under Ex. 488 nm and Em. 530-560 nm (Channel 1 (C1)) and Ex. 561 nm and Em. 570-600 nm (Channel 2 (C2)). Stacks of 0.33 μm steps were obtained. A straight line was drawn from the base of the PHCs until the tip and plot profiles of gray values for C1 and C2 (same defined ROI) were obtained from the sum slices Z-projections. The ratio between the plot profiles from C2 and C1 was obtained. Additionally, images were analyzed using the Fiji macro developed by Baldacci-Cresp et al. 2020 for calculating a ratio from a generated ratiometric image (C2/C1) 37 .

Statistical Analysis
To test for significant differences between two groups we performed unpaired t-tests. One-way ANOVAs and multiple comparison tests were used when comparing more than two groups. Significance was determined when the p value was lower than 0.05. p values are indicated directly in the graphs and details on each analysis described in the figure legends of the respective graphs. All analyses were performed on GraphPad Prism version 9.1.0, GraphPad Software, San Diego, CA, USA, www.graphpad.com.